Both cell-cell and cell-matrix adhesions require the participation of the actin cytoskeleton. Actin filaments are crosslinked into circumferential rings that are associated with cell-cell junctions and contractile stress fibers that are anchored at cell-matrix junctions. Palladin is a recently identified protein that localizes to cell-cell and cell-matrix junctions, and also to stress fibers, Z-discs and other actin-rich structures. In addition to cell culture studies that demonstrate palladin's essential role in the assembly of the actin cytoskeleton, two other lines of evidence suggest that palladin plays a central role in actin-dependent cell behaviors: (1) palladin is critically important for normal mammalian development, as evidenced by the embryonic lethal phenotype of the palladin knockout mouse and (2) the human palladin gene is mutated in a form of inherited pancreatic cancer, which is a disease characterized by a loss of cell-cell adhesion and disregulated, actin-dependent, invasive cell motility. The goal of this proposal is to understand the precise molecular function of palladin in organizing actin arrays;specifically, we will test the hypothesis that palladin functions as an actin-crosslinking protein, both in vitro and in vivo. Palladin exists as three major isoforms, containing between three and five copies of a highly conserved Ig-like domain. Published studies of three other Ig-domain proteins suggest that palladin's Ig domains may bind directly to f-actin. We have generated preliminary results showing that purified, full-length palladin binds to f-actin and generates actin bundles in vitro. In addition, our results show that the domain designated Ig3 is the smallest palladin fragment that binds to actin, while Ig4 and Ig5 do not bind, and that a fragment containing the Ig3+Ig4 domains is able to cross-link actin filaments into bundles. To fully investigate the molecular basis for this effect, we propose to express, purify and characterize all of the Ig domains of palladin. Our aims are (1) to conclusively map the actin binding site contained within palladin's Ig domains, using our recently determined solution structure of Ig3 and rational mutagenesis approaches, and to investigate the role of actin-binding in palladin's biological activity, (2) to test the hypothesis that palladin functions as an actin- crosslinking protein in vitro and in vivo, and to determine the geometry of the actin-generated bundles by both fluorescence and electron microscopy, and (3) to test the hypothesis that palladin forms homodimers, map the dimerization domains, and establish if dimer formation is key to palladin's biological function. In addition, we will determine the structure of palladin's Ig4 domain and investigate a novel mutation in this domain that has been identified in a pancreatic cancer cell line. To date, no structural information has been published regarding the Ig domains of palladin or palladin's close family members, such that our proposed research represents the first detailed structure/function analysis of palladin's Ig domains. Public Health Relevance: Actin filaments form complex subcellular arrays that are essential for establishing cell shape, maintaining adhesion between neighboring cells, and generating connections between cells and their underlying connective tissue. Palladin is a recently described protein that plays a key role in organizing these actin filament arrays, and mutation or over-expression of palladin has been implicated in multiple pathologies, including lethal birth defects, scar formation in the skin, vasculature and nervous system, and the invasion of pancreatic cancer. Thus, understanding the precise molecular function of palladin is likely to yield insights into the cellular mechanisms that control both essential functions in normal cells, and dysfunctions that result in pathological cell behavior.